An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abaca- vir (ABC), 20,30-dideoxyinosine (DDI), lamivu-dine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegra- vir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37°C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37°C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV>3TC>AZT>NVP>DDI> ABC and ETR>RPV>RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the opti- mized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quanti-tative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.
Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3
Mastino Antonio;
2016
Abstract
An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abaca- vir (ABC), 20,30-dideoxyinosine (DDI), lamivu-dine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegra- vir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37°C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37°C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV>3TC>AZT>NVP>DDI> ABC and ETR>RPV>RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the opti- mized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quanti-tative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
